There is a balance between factors promoting renal salt and water retention and those favoring excretion. Inappropriate salt retention may lead to hypertension. This project focuses on superoxide (O2-), which favors salt and water retention. Renal O2- is an important regulator of kidney function and has been implicated in salt-sensitive hypertension. The thick ascending limb of the loop of Henle is one of the nephron segments responsible for inappropriate NaCI retention in salt-sensitive hypertension and is the site of action of loop diuretics. In the last funding period we showed that O2- increased thick ascending limb transport by enhancing the activity of the luminal Na/K/2CI cotransporter. This effect was due to both activation of protein kinase C (PKC) and scavenging of nitric oxide (NO). However, few studies have directly addressed how O2- production is regulated along the nephron. Urine flow through thick ascending limbs is not static, but acutely varies from more than 25 nl/min to as little as 0 nl/min (actually stopping) due to peristalsis of the papilla. Our preliminary data show that increasing luminal flow through the thick ascending limb (THAL-flow) stimulates O2- generation. Our general hypothesis is that factors that increase urine flow and therefore augment stretch, pressure or shear stress in the thick ascending limb enhance O2- production, which in turn promotes NaCI absorption and therefore Na retention. The increase in Na retention caused by enhanced O2- in the thick ascending limb may be important in the pathogenesis of hypertension and other diseases associated with Na retention. Aim 1 will test whether acutely increasing THAL-flow causes the assembly and activation of NADPH oxidase, thereby enhancing O2- generation and NaCI absorption. Aims 2 and 3 will study the signaling cascades responsible for flow-stimulated O2- production focusing on protein kinase C and the small G-protein Rac1. Aim 4 will investigate how flow-induced NO regulates NADPH oxidase activity and its impact on NaCI absorption. We will employ state of the art techniques to address these aims, including fluorescence energy transfer and in vivo viral transduction in addition to standard physiological, biochemical and pharmacological techniques. Successful completion of this project will give us new insights into how O2- is generated in the kidney as well as its role in salt retention, and predict new targets for treatment of hypertension.